【作者】 陶杰；

【导师】 韩雅玲； 闫承慧；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 硕士

【摘要】 目的经皮冠状动脉介入治疗(PCI)术后支架内再狭窄(ISR)以及支架内迟发性血栓形成是心血管介入界尚未攻克的难题,内皮细胞(ECs)的损伤及功能障碍是ISR的始动因素和迟发性血栓形成的重要原因,促进ECs的修复成为防治ISR和迟发性血栓的一个热点。E1A激活基因阻遏子(CREG)是新近发现的一个细胞转录调控基因,其在分化成熟的组织、细胞中广泛表达,而在去分化的组织中低表达。已有研究表明CREG可促进体外培养的人血管平滑肌细胞(VSMCs)分化成熟,抑制其增殖、凋亡,说明CREG对VSMCs的稳态起着调控作用,具有对抗ISR发生的潜在价值。而CREG对动脉ECs的生物学效应尚未见报道。本研究以体外培养的人动脉ECs为研究对象,应用逆转录病毒载体介导CREG在动脉ECs中过表达及表达下调,观察CREG表达在动脉ECs迁移、增殖中的作用,并初步探讨其作用机制。方法(1)用脂质体转染法将重组人CREG逆转录病毒表达载体pLNCX-CREG、CREG干扰RNA逆转录病毒表达载体pLNCX-shRNA-CREG及带绿色荧光蛋白(GFP)的空载体pLNCX-GFP转染phoenix 293细胞,包装出完整的逆转录病毒后,感染动脉ECs,分别经G418和嘌呤霉素筛选,挑取单细胞克隆培养,获得稳定感染的细胞克隆。将细胞分为4组进行实验:正常组、GFP转染对照组、CREG过表达组和CREG表达下调组。(2)RT-PCR检测抗性基因表达;Western blot检测感染前后动脉ECs中CREG蛋白表达。(3)刮伤试验、Transwell迁移模型观察感染前后动脉ECs迁移的变化,明胶酶谱分析检测明胶酶活性改变。(4)细胞计数、流式细胞分析检测感染前后ECs的增殖改变。(5)Western blot检测信号通路蛋白PI3K、p-Akt、p-JNK、p-ERK、p-p38的表达以及与ECs迁移、增殖和CREG蛋白调控相关的尿激酶型纤溶酶原激活剂(u-PA)、内皮细胞型一氧化氮合成酶(eNOS)、血管内皮生长因子(VEGF)的表达;ELISA检测培养上清中VEGF、胰岛素样生长因子Ⅱ(IGFII)的分泌。(6)在各组细胞中分别添加LY 294002、SP 600125、PD 98059和SB 203580以抑制P13K-Akt、JNK-MAPK、ERK-MAPK和p38-MAPK信号通路,应用Transwell迁移模型观察各组细胞迁移的改变;在各组细胞中添加上述信号通路阻断剂,流式细胞分析检测各组细胞增殖的改变。(7)在过表达CREG的ECs中添加重组人IGFII(rhIGFII)、重组人IGFII受体(rhIGF2R)和anti-IGF2R中和抗体;在CREG表达下调组细胞中添加重组人VEGF(同时添加或不添加VEGF中和抗体),应用Transwell迁移模型观察不同处理组细胞迁移的变化,探讨IGFII、VEGF在CREG调控动脉ECs迁移中的作用。结果(1)将重组逆转录病毒表达载体pLNCX-GFP、pLNCX-CREG及pLXSN-shRNA-CREG用脂质体法分别感染phoenix 293细胞,包装生产病毒,感染人动脉ECs,经过G418及嘌呤霉素筛选、挑取单克隆,分别得到表达GFP、CREG过表达及表达下调的动脉ECs克隆。(2)感染细胞的分别表达G418或嘌呤霉素抗性基因;Western blot结果显示,与正常ECs及GFP感染组ECs比较,pLNCX-CREG逆转录病毒感染组ECs中CREG蛋白表达上调;而感染pLNCX-shRNA-CREG逆转录病毒的ECs中CREG蛋白表达明显下调。(3)CREG过表达促进ECs迁移。a.刮伤实验及Transwell迁移模型提示:与GFP对照组和未感染细胞相比,CREG过表达促进动脉ECs迁移,而CREG表达下调抑制动脉ECs迁移;b.明胶酶谱分析提示:与对照组相比,CREG表达下调组明胶酶明显减低;而CREG过表达组与对照组相比呈增加趋势,但无明显差异。(4)细胞计数及流式细胞分析结果表明:CREG过表达的动脉ECs增殖能力增强,而CREG表达下调组增殖能力减弱。(5)a.Western blot结果显示:与对照组相比,PI3K/Akt信号转导蛋白表达在CREG过表达组增加,而在CREG表达下调组减少;MAPK信号转导通路中p-ERK、p-JNK、p-p38均无明显变化。b.Western blot结果提示:与对照组相比,u-PA、eNOS和VEGF在CREG过表达组ECs中表达增加,在CREG表达下调组表达减低。c.ELISA结果提示:CREG过表达ECs培养液中VEGF分泌增多,IGFII分泌减少,而CREG表达下调组VEGF分泌减少,IGFII分泌增多。(6)a.Transwell迁移模型的信号通路阻断研究结果提示:CREG过表达通过PI3K/Akt信号转导通路促进动脉ECs迁移;b.流式细胞分析的信号通路阻断剂研究结果提示:PI3K/Akt、ERK-MAPK、JNK-MAPK信号通路都不同程度参与了ECs的增殖过程,但PI3K/Akt信号转导通路介导CREG调控的ECs增殖。(7)a.在CREG过表达的ECs Transwell迁移模型中添加rhIGFII生长因子以增加细胞上清中IGFII的量,或者添加rhIGF2R小肽片段和IGF2R中和抗体以阻断IGF2R介导的IGFII内吞后,均明显抑制CREG过表达介导的细胞迁移增加;b.在CREG表达下调的ECs Transwell迁移模型中添加重组人VEGF可明显促进细胞迁移;但同时添加VEGF和anti-VEGF中和抗体,可消除VEGF对ECs迁移的促进作用。上述结果提示CREG过表达可能通过促进VEGF的分泌和IGFII的内吞部分介导了动脉ECs迁移。结论(1)CREG过表达可促进体外的培养ECs的迁移和增殖。相反,CREG表达下调抑制了ECs的迁移和增殖;(2)PI3K/Akt信号转导通路表达及活化介导了CREG对ECs增殖和迁移的调控;(3)CREG过表达可能通过促进ECs中VEGF的分泌及IGFII的内吞,部分介导了其对ECs迁移的调控。本工作提示CREG的表达有助于促进ECs迁移与增殖,促进血管稳态的维持,为PCI术后ISR及迟发性血栓的防治提供了新的思路。更多还原

【Abstract】 Objective:In-stent restenosis(ISR)after percutaneous coronary intervention (PCI)and late stent thrombosis are two major issues unresolved in the field of interventional cardiology.Endothelial injury and dysfunction of coronary arteries were regarded as an initiation factor for restenosis and were also found playing essential roles in formation of late stent thrombosis.So research on promoting endothelial repair will potentially provide novel targets for both prevention and treatment of ISR and late stent thrombosis.The cellular repressor of E1A stimulated genes(CREG)was a transcriptional regulator found by Gill in 1998,which expressed at high level in the differentiated and mature tissues and cells in contrast to at low level in the undifferentiated and immature ones.Moreover,our lab has identified that CREG could promote differentiation of human vascular smooth muscle cells(VSMCs)and inhibit their proliferation and apoptosis in vitro.These findings suggested that CREG is an important endogenous factor in regulating VSMCs homeostasis and have promising utility in strategy of anti-restenosis.Although CREG has been extensively investigated in VSMCs,there is no reported research exploring roles of CREG in regulating function of arterial endothelial cells(ECs).In this study,human arterial ECs were tansfected with retroviral eukaryotic vectors, and their expression of CREG were up-or down-regulated.The aim of this study was to investigate the effects of CREG on ECs migration and proliferation,and further illustrate their underlying mechanisms.Methods:(1)Phoenix 293 cells were transfected with sense-CREG retroviral vector(pLNCX-CREG),RNA interference CREG retroviral vector(pLNCX-shRNA -CREG)or green fluorescent protein(GFP)expressing control retroviral vector(pLNCX-GFP)by liposome method respectively.Then ECs were infected with packaged viruses collected from transfected phoenix 293 cells. Single ECs clones stably infected with different viruses were selected with corresponding antibiotics(G418 or puromycin).ECs were divided into 4 groups according to different viruses they are infected:uninfected normal control ECs(group EN);GFP expression control ECs(group EG);CREG overexpression ECs(group EO)and CREG supression ECs(group ES).(2) Antibiotic genes expression was detected through RT-PCR and CREG expression of ECs before or after its being infected by retrovirus was detected Western blot.(3)Effects of CREG on ECs migration were investigated by scrape injury analysis and Transwell migration model.Zymography was performed to investigate the change of gelatinases.(4)Cell counting and flow cytometry were performed to detect the ECs proliferation in different groups. (5)Expression of signaling pathway proteins including PI3K,p-Akt,p-TNK,p-p38, p-ERK were detected through Western blot in all groups.Also,Expression of urokinase plasminogen activator(u-PA),vascular endothelial growth factor (VEGF)and endothelial nitric oxide synthase(eNOS)involved in cell migration or proliferation were examined by Western blot analysis.The secretion of VEGF and IGFⅡin the medium was tested by Enzyme linked immunosorbent assay(ELISA).(6)In Transwell migration model and flow cytometry assay, changes of ECs migration or proliferation were observed after administration of corresponding inhibitors of signal protein including LY 294002,SP 600125, PD 98059 and SB 203580,which inhibited the PBK-Akt,JNK-MAPK,ERK-MAPK or p38-MAPK signal pathway respectively.(7)Recombinant human IGFII(rhIGFII),rhIGF2R or anti-IGF2R neutralizing antibody was added to the culture medium of CREG overexpression ECs solely in Transwell migration model.Also,solely or accompanied with anti-VEGF neutralizing antibody,VEGF was added to the culture medium of CREG suppression ECs. The ECs migration was accessed in both groups.Results:(1)Three kinds of retroviruses,packaged by phoenix 293 cells transfected with pLNCX-GFP,pLNCX-CREG and PLXSN-shRNA-CREG vectors,were used to infect ECs and after selection with G418 or puromycin, single cell clones were picked up and replanted for amplification.The ECs wih GFP expression,CREG overexpression or suppression were obtained.(2)The tansfected ECs clones transcripted G418 or puromycin resistance genes respectively,which was identified by RT-PCR assay.Western blot analysis showed that CREG expression increased in group EO(infected by pLNCX-CREG retrovirus),decreased obviously in group ES(infected by pLNCX-shRNA -CREG retrovirus)compared to group EN.(3)Overexpression of CREG promoted ECs migration.a.The scrape injury assay and Transwell migration model suggested that CREG overexpression enhanced but its suppression attenuated migration of ECs compared to the normal and GFP expression control groups ECs.b.Gelatin zymography indicated that expression of gelatin rose in CREG overexpression ECs compared to CREG suppression ECs,but it made no statistics difference in comparison with normal and GFP expression groups.(4)The cell count and Flow cytometry assay results showed that proliferation of ECs was enhanced in CREG overexpression group and attenuated in the CREG suppression group.(5)a.Western blot results suggested that protein expressions of PI3K and p-Akt signal pathway increased in the CREG overexpression group ECs and decreased in the CREG suppression group ones,but there was no difference between the expression of p-JNK,p-p38 and p-ERK protein involved in the MARK signal pathway.b.Also,the expression of u-PA,eNOS and VEGF was augmented in the CREG overexpression group ECs and down regulated in the CREG suppression group ECs.c.ELISA assay suggested that secretion of VEGF increased but that ofⅠGFⅡdecreased in the CREG overexpression group compared with that in the control groups ECs,and the opposite results was found in CREG suppression group ECs.(6)a. Transwell migration model showed that CREG promoted the ECs migration through the PI3K/Akt signal transduction pathway.b.Flow cytometry indicated that PI3K/Akt,ERK-MAPK,JNK-MAPK signal pathways were involved in the regulation of proliferation of ECs,but promotion of ECs proliferation induced by CREG overexpression was mediated through the PBK/Akt signal pathway.(7)a. Addition of rhⅠGFⅡ,rhⅠGF2R peptide fragment or anti-ⅠGFⅡR neutralizing antibody repress cells migration mediated by CREG overexpression.b.The migration of CREG suppression ECs was promoted by addition of recombinant human VEGF in its culture medium,which can be eliminated by the accompanied addition of anti-VEGF neutralizing antibody.These results indicated that overexpression of CREG can possibly promote artery ECs migration partly by the way of regulation of secretion of growth factors,such as VEGF andⅠGFⅡ.Conclusions:(1)Overexpression of CREG can promote the migration and proliferation of ECs in vitro.(2)PI3K-Akt signal pathway mediated the overexpressing CREG promotion on ECs migration and proliferation.(3)The artery ECs migration was promoted by CREG overexpression partly through its regulating the secretions of VEGF andⅠGFⅡ.Our study showed that CREG facilitates migration and proliferation of ECs and contributes to the maintenance of vascular homeostasis.This may provide a promising approach for the prevention and treatment of ISR and late stent thrombosis after PCI.更多还原